Fluorescence is often used in biology and particularly in optical microscopy for tissue identification. A fluorophore chosen for its abilities to bind to or ‘tag’ a specific type of tissue is introduced into the sample being interrogated. When the sample is illuminated by light at the excitation wavelength of the fluorophore, the tissue tagged by the fluorophore will emit light at the fluorophore's emission wavelength, thereby allowing optical detection of that tissue's presence and evaluation of the tissue's distribution within the sample.
A sizeable industry exists to support fluorescent optical microscopy. Several microscope manufacturers (e.g. Nikon Zeiss, Olympus) offer adapters for their instruments that allow fluorescent microscopy using the microscopes' existing light sources. Biochemical suppliers such as Fluka BioChemika, Molecular Probes, Fuji Photofilm and Sigma-Aldrich supply scores of different fluorophores that are designed with specific optical and biochemical properties. Optics companies such as Omega Optical, Barr Associates, and Semrock supply optical filters for both the excitation and fluorescent emission wavelengths, allowing customization of microscopy equipment for specific fluorophores.
An extension of the concept of fluorescent microscopy is to image tissue-bound fluorophores in animals in vivo, or immediately post mortem, to ascertain the distribution of the tagged tissue. In this application, typically a white-light source is filtered with an excitation filter appropriate to the fluorophore, and a video camera, usually a CCD camera, views the test subject through an appropriate fluorescence emission filter. The camera produces a two-dimensional image that is a projection of the fluorophore's distribution onto a plane, much like a conventional two-dimensional x-ray. Although these two-dimensional images show the distribution of the fluorophore, accurate estimation of the quantity of tagged tissue within the sample (e.g. the volume of a tumor) is difficult without using actual three-dimensional fluorescent images of the sample. Several companies produce 2-D fluorescence imaging systems, notably Xenogen's IVIS® Imaging System, Berthold Technologies' NightOWL LB98, and ART Advanced Research Technologies, Inc.'s SAMI. None of these systems, however, show volumetric data that can allow quantitative estimation of tissue volumes.
Prior art fluorescent imaging systems, such as those described above, are used in cancer research to evaluate anticancer treatments in nude mice. These hairless mice carry a recessive gene that inhibits the development of the thymus gland. The mice are unable to generate mature T-lymphocytes and therefore are unable to mount most types of immune responses, including antibody formation and rejection of transplanted tissues. Cancerous allografts and xenografts are readily accepted and nurtured by the mice, making them excellent vehicles for the study of human cancers and their reactions to different treatments. Treatment efficacy is monitored by injecting a tumor-bearing nude mouse with a cancer-specific fluorophore and then tracking the change in tumor size with a fluorescent imaging system.
A technique developed in the mid-1990's creates cancer cells that are genetically altered to fluoresce, obviating an injected fluorophore. Green fluorescent protein (GFP), which is produced by certain jellyfish, emits green light when exposed to certain wavelengths of blue light. By ‘transfecting’ the appropriate DNA segment from these jellyfish into other cells, the cells are made to express GFP. In cancer research, the cancer cells are transfected with the GFP gene, and the progression of the tumor or its metastases can be monitored noninvasively by its GFP fluorescence using fluorescence imaging.
Recently, cells have been genetically modified to express fluorophores at other wavelengths (particularly by the Clontech division of Becton Dickinson), but GFP-expressing cells are much more widely used than any of these new cells.
Human cancer cells are being grown that are transfected with the GFP gene. These cancer cells are transplanted into nude mice, which do not reject the cancer, rather they nourish the cancer, allowing testing of anticancer treatments. The progression of the cancer or its metastases can be imaged with fluorescence of the GFP.